pREM: a positive selection vector system for direct PCR cloning

Abstract

The present invention describes the development of a positive selection vector based on regulatory element modulation, wherein such modulation is achieved via insertional reconstruction or destruction of a regulatory element controlling transcription, translation, DNA replication and termination. A positive selection cloning vector pREM5Tc has been developed based on insertional reconstruction of a regulatory element of a reporter gene. The vector pREM5Tc carries the tetracycline resistance reporter gene with no functional -35 region of its promoter, a regulatory element, thus resulting in no expression of the tetracycline resistance gene. Hence a host cell carrying the vector pREM5Tc is unable to produce the tetracycline resistance gene protein resulting in inhibition of its growth in presence of tetracycline. An E. coli consensus -35 region is recognized as 5'-TTGACA-3' and a primer used in polymerase chain reaction (PCR) carries at its 5' end the sequence 5'-TGTCAA-3', which is the complementary sequence of 5'-TTGACA-3'. The PCR-amplified DNA fragment is ligated to pREM5Tc thus reconstructing the functional promoter of the tetracycline resistance reporter gene. Subsequent transformation of a host cell with the recombinant vector (carrying an insert DNA) results in production of the tetracycline resistance reporter gene protein that confers resistance to tetracycline thus allowing only the recombinants to grow in presence of tetracycline. The positive selection vector pREM5Tc greatly reduces, if not eliminates, the number of exonuclease-generated false positive clones.

Description

OTHER REFERENCESAhrenhotz et al., "A conditional suicide system in Escherichia coli based on intracellular degradation of DNA" Appl. Environ. Microbiol. 60,3746-3751(1994).Altenbuchner et al., "Positive selection vectors based on palindromic DNA sequences" Methods Enzymol. 216, 457-466 (1992).Balbas et al., "Plasmid vector pBR322 and its special-purpose derivatives--a review" Gene 50, 3-40 (1986).Bernard et al., "New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers" Gene 148, 71-74 (1994).Bolivar et al., "Construction and characterization of new cloning vehicles, II. A multipurpose cloning system" Gene 2, 95-113 (1977).Burns D. M. and Beacham, I. R., "Positive selection vectors: a small plasmid-vector useful for the direct selection of Sau3A-generated overlapping DNA fragments" Gene 27, 323-325 (1984).Clark, J. M., "Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases" Nucl. Acids Res. 1...

AI Technical Summary

Benefits of technology

The object of the present invention is to develop a simple cloning and/or a sequencing vector which should have the capability of positive selection allowing only the recombinant clones (carrying an insert DNA) to grow in a selection medium, whereas, the non-recombinant clones (carrying no insert) will not grow. A major object of the present invention is to eliminate or greatly reduce the false positive clones associated with all the presently available cloning systems. Especial emphasis is placed on the elimination of exonuclease-induced false positive clones. Thus it is aimed to apply the principle of modulation of a regulatory element, which involves in

Problems solved by technology

When the number of recombinant colonies are low and non-recombinant colonies are high in a plate, then it becomes very difficult to differentiate the recombinant colonies from the non-recombinant colonies.

High number of colonies on a plate also lead to contamination between the recombinant and non-recombinant colonies.

An inherent problem of a vector with a lethal or a chromogenic gene is a high number of false positive clones, i.e., clones without any insert.

However, the biggest disadvantage of every cloning system available today is the generation of exonuclease-induced false positive clones.

The reagents used in restriction digestion, PCR and ligation, particularly restriction enzymes, polymerases and ligases, are

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